The molecular effect of domestication has rarely been examined in fungi. I examined the molecular signature of domestication on Aspergillus oryzae, a fungus used to ferment several Japanese food products. The ratio of nonsynonymous to synonymous substitutions and the percentage of estimated deleterious substitutions along the A. oryzae lineage were lower than those in the wild sister lineage Aspergillus flavus. The patterns of genetic change observed in human-mediated domestication of animals and plants might not be typical in domesticated fungi.